Our major research aim is to clarify how interactons between embryonic tissues lead to cell determination, and to understand how changes in gene expression result from these tissue interactions. This proposal is concerned with the latter issue, which is addressed by examining how the genes encoding the major lens-specific protein in the chick, delta-crystallin, are regulated during lens development. First, precise time course studies will be performed to analyze the activation of data-crystallin genes in the lens lineage. This system is particularly advantageous for such a developmental study since lens or presumptive lens cells can be isolated before and during commitment to the lens cell type, and subsequently, when delta-crystallin is first synthesized, as well as at all later stages. Experiments are planned to determine when the delta-crystallin genes are first transcribed during lens development and whether alteration in chromatin structure or DNA methylation occur before, during or after this gene activation. Our second goal is to begin an analysis of how inductive signals lead to the regulation of gene expression in this system. This question will be examined in ventral or lateral head ectoderm which is some distance from the normal lens. This ectoderm itself has the potential to form a lens if cultured alone, though it is normally prevented from doing so by an inhibitory signal from the mesoderm underlying it. Experiments will ask whether this inhibitory interaction has any direct effect on the delta-crystallin genes, by preventing methylation changes, inhibiting the appearance of DNase I hypersensitive sites, or perhaps preventing processing of delta-crystallin transcripts. Finally, the lens system will be used to investigate how hypomethylation and DNase I hypersensitivity occur, particularly whether DNA replication is integral for either process. Experiments will distinguish two possible models for how hypomethylation might occur, either by demethylation or by simply not methylating newly replicated DNA. It is important to establish whether DNase I hypersensitive sites in chromatin appear only on newly synthesized DNA, since this would imply that chromatin remodeling is associated with DNA replication.